Obesity and diabetes lead to high circulating levels of insulin and high blood pressure (BP). However, mechanisms underlying these associations are in dire need of clarification. For example, in the kidney, insulin, through its own receptor, can lead to sodium retention, and activate the epithelial sodium channel (ENaC). Nonetheless, insulin can also increase the production of nitric oxide (NO), which has been shown to reduce the activity of ENaC. Thus, these two actions of insulin would be expected to have opposing effects on BP. To better elucidate the role of the insulin receptor in the kidney, we have developed an transgenic mouse with knockout (KO) or deletion of the insulin receptor (IR) in the distal tubule of the kidney (IRKO). These mice survive and grow normally, but have significantly higher basal BP. They are also impaired in their ability to rapidly excrete a NaCl load, as well as, have blunted rise in urinary nitrates plus nitrites (a urinary form of NO) excretion in the basal state and after insulin treatment. Thus our hypothesis is that IR in the kidney may have a previously unappreciated role in facilitating volume excretion and in the maintenance of normal BP. We further suggest these deficiencies arise directly as the result of impaired NO production with subsequent over-activity of ENaC. Three main aims are outlined below. Aim 1 is to determine whether reduced renal NOS activity in the distal tubule is a mechanistic determinant of the sodium-excretory defect and elevated BP in the IRKO mice. Renal nitric oxide synthase (NOS) activity will be measured in inner medulla, outer medulla, and cortex, as well as, microdissected proximal tubule, thick ascending limb, and collecting duct from fed and fasted IRKO and WT mice. We will also test the direct effects of insulin on NOS activity and NO levels in inner medullary collecting duct (IMCD) cultures. Finally, we will test whether molsidomine, an NO donor, restores NO levels, as well as normalizes BP and sodium excretion in IRKO mice. Aim 2 is to determine whether increased activity of the epithelial sodium channel (ENaC) is a mechanistic determinant of the sodium-excretory defect and elevated BP in the IRKO mice. For this aim, we will test whether benzamil, an antagonist of ENaC, abolishes differences in BP and natriuresis. ENaC regulation will be examined in native tissue and in primary IMCD cells. Finally, in Aim 3 we determine whether distinct IR signaling relating to ENaC activation and NO generation is altered in the IRKO mouse IMCD. Phosphorylation of critical proteins involved in IR signal transmission from the receptor to the activation of ENaC and nitric oxide synthase will be evaluated. These proteins include, but are not limited to, the insulin receptor substrate (IRS), phosphoinositide-3-kinase (PI-3K), and the serum and glucocorticoid-regulated kinase (SGK1). Overall, these studies will highly elucidate the role of insulin in the the distal tubule with regard to its role in blood control and sodium regulation. Moreover, they may provide insight into the pathology underlying hypertension associated with the metabolic syndrome. PUBLIC HEALTH RELEVANCE: Insulin resistance is associated with high circulating plasma levels of insulin, as well as, increased blood pressure; however, cellular mechanisms relating the two are not well understood. The kidney, a major regulator of blood pressure, expresses insulin receptors along the renal tubule; however, their role there is undefined. These studies are aimed at elucidating insulin's role in the distal tubule, which will hopefully allow for earlier and better targeted treatments in obesity and diabetes.